U.S. patent application number 16/330020 was filed with the patent office on 2019-07-04 for aqueous extract of prunus persica and process for preparing the same.
This patent application is currently assigned to ISP Investments LLC. The applicant listed for this patent is ELC Management LLC, ISP Investments LLC. Invention is credited to Jean-Marie BOTTO, Nouha DOMLOGE, Isabelle IMBERT, Dawn LAYMAN, Audrey LE MESTR, Elodie OGER, Nadine PERNODET.
Application Number | 20190201321 16/330020 |
Document ID | / |
Family ID | 59895404 |
Filed Date | 2019-07-04 |
United States Patent
Application |
20190201321 |
Kind Code |
A1 |
PERNODET; Nadine ; et
al. |
July 4, 2019 |
AQUEOUS EXTRACT OF PRUNUS PERSICA AND PROCESS FOR PREPARING THE
SAME
Abstract
The present invention provides a process for obtaining an
extract from peach flower extract (Primus persica L.) for skin
cosmetic use, a peach flower extract obtainable by the process and
a cosmetic composition comprising the said extract. The invention
further provides a method for modulating the SIRT2 expression, a
method of treatment designed to reduce and/or correct the signs of
aging and photo-aging of the skin and keratinous appendages, and to
protect the skin against aggressions due to ultraviolet
radiation.
Inventors: |
PERNODET; Nadine;
(Huntington Station, NY) ; LAYMAN; Dawn; (Ridge,
NY) ; BOTTO; Jean-Marie; (Valbonne, FR) ;
OGER; Elodie; (Vallauris, FR) ; LE MESTR; Audrey;
(Antibes, FR) ; IMBERT; Isabelle; (CANNES, FR)
; DOMLOGE; Nouha; (Opio, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
ISP Investments LLC
ELC Management LLC |
Wilmington
Melville |
DE
NY |
US
US |
|
|
Assignee: |
ISP Investments LLC
Wilmington
DE
ELC Management LLC
Melville
NY
|
Family ID: |
59895404 |
Appl. No.: |
16/330020 |
Filed: |
September 6, 2017 |
PCT Filed: |
September 6, 2017 |
PCT NO: |
PCT/US2017/050223 |
371 Date: |
March 1, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62384436 |
Sep 7, 2016 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 2800/70 20130101;
A61K 8/347 20130101; A61K 8/60 20130101; A61K 2800/805 20130101;
A61Q 17/04 20130101; A61Q 19/08 20130101; A61K 8/64 20130101; A61K
8/9789 20170801; A61K 8/97 20130101 |
International
Class: |
A61K 8/9789 20060101
A61K008/9789; A61Q 19/08 20060101 A61Q019/08; A61Q 17/04 20060101
A61Q017/04 |
Claims
1. A process for obtaining an extract from peach flower (Prunus
persica L.) comprising: (i) adding water to peach flower to make a
mixture, (ii) agitating the said mixture for 2 hours by maintaining
temperature from RT to below 70.degree. C. (iii) filtering the
mixture to remove the solid flower part to obtain the extract. (iv)
pasteurizing the extract overnight at a temperature below
70.degree. C.
2. The process according to claim 1, wherein said peach flower is
pink in colour.
3. The process according to claim 1 wherein step (ii) is made at a
temperature ranging from RT to 50.degree. C.
4. The process according to claim 1 wherein step (ii) is made at a
temperature of 50.degree. C.
5. The process according to claim 1, wherein said peach extract is
obtained from dried peach flower.
6. The process according to claim 1, wherein said extract of step
(iv) is further clarified by sequential filtration from about 50
.mu.m to about 20 .mu.m porosity until about 0.5 .mu.m to about 0.2
.mu.m to get the extract.
7. The process according to claim 1, wherein said peach flower
extract of step (iv) is then diluted in a solvent selected from
water, glycerol, ethanol, propanediol, butylene glycol, dipropylene
glycol, ethoxylated or propoxylated glycols, cyclic polyols or any
mixture of these solvents.
8. A peach flower extract obtained by the process of claim 1.
9. The peach flower extract of claim 8 comprising compounds having
a molecular weight of less than 10 kDa.
10. The peach flower extract of claim 8, wherein DPPH free
radical-scavenging activity is less than 80 mg/L.
11. The peach flower extract of claim 8, wherein said peach flower
extract comprises from 8 to 15 g/kg of dry matter, from 2 to 8 g/L
of protein, from 2 to 8 g/L of sugar, from 0.2 to 2 g/L of amino
acids; and from 0.2 to 2 g/L of phenolic compounds.
12. The peach flower extract of claim 8, wherein the phenolic
compounds comprise caffeic acid, catechin, rosmarinic acid, gallic
acid, coumaric acid and hydroxybenzoic acid.
13. A cosmetic composition comprising a peach flower extract
obtained according to the process of claim 1, wherein the said
peach flower extract comprises compounds having a molecular weight
of less than 10 kDa and a physiologically acceptable medium.
14. The cosmetic composition of claim 13, wherein said peach flower
extract is used in a concentration from about 0.0001% by weight to
about 20% by weight of the total weight of the composition.
15. The cosmetic composition of claim 13, wherein said peach flower
extract is used in a concentration from about 0.0005% by weight to
about 5% by weight of the total weight of the composition.
16. The cosmetic composition of claim 13, wherein said composition
is in a form adapted for topical application.
17. The cosmetic composition of claim 13, wherein said composition
is in the form of gels, creams, balms, milks or foaming
products.
18. The cosmetic composition of claim 13, further comprising at
least one other active agent.
19. The cosmetic composition of claim 13, further comprising at
least one other active agent selected from antioxidant agent,
hydrolysate of plants, synthetic peptide compound, sunscreen and
anti-wrinkle agent.
20. A method for reducing and/or correcting the signs of aging and
photo-aging of the skin and keratinous appendages, comprising
topically applying to the skin, the mucous membranes and/or
superficial skin appendages a cosmetic composition comprising the
peach flower extract obtained according to the process of claim 1,
wherein the peach flower extract comprises compounds having a
molecular weight of less than 10 kDa in a physiologically
acceptable medium.
21. The method of claim 20 to protect the skin against aggressions
due to ultraviolet radiation, comprising applying topically on the
skin to be treated a cosmetic composition.
22. A method for modulating the SIRT2 expression, comprising
topically applying to the skin, the mucous membranes and/or
superficial skin appendages a cosmetic composition comprising the
peach flower extract obtained according to the process of claim 1,
wherein the peach flower extract comprises compounds having a
molecular weight of less than 10 kDa in a physiologically
acceptable medium.
Description
TECHNICAL FIELD
[0001] The present invention is in the field of cosmetics and more
specifically in the field of skin care. The invention relates to a
process for preparing a peach flower (Prunus Persica) extract by
aqueous extraction, a peach flower extract obtainable by the
process and a cosmetic composition comprising the peach flower
extract.
[0002] The invention also relates to a method for modulating the
SIRT2 expression, to a cosmetic method of treatment designed to
reduce and/or correct the signs of aging and photo-aging of the
skin and keratinous appendages, and to protect the skin against
aggressions due to ultraviolet radiation.
[0003] The Peach flower extract can be used alone or in combination
with other active agents.
BACKGROUND
[0004] The skin is a vital organ composed of several layers
(dermis, proliferative layers and stratum corneum), which covers
the entire surface of the body and ensures protective, sensitive,
immune, metabolic or thermoregulatory functions. The skin, like the
other organs, is subject to aging.
[0005] For example, the appearance of the skin is modified by
various types of internal (disease and hormonal changes such as
pregnancy) or external aggressions (environmental factors, such as
pollution, sunlight, pathogens, etc.). Then wrinkles and fine
lines, hyperpigmentation or hypopigmentation blemishes, dryness or
even dehydration of the skin, thinning of the epidermis, elastosis,
imperfections, age spots, etc., may appear. All of these changes
affect not only the skin, but also the keratinous appendages such
as the nails and hair.
[0006] It is known that free radicals, chemically unstable and very
reactive species generated by the intracellular metabolism or
external aggressions, play a key role in the aging process and more
particularly in the formation of oxidized, damaged proteins (Harman
et al. "Aging: a theory based on free radical and radiation
chemistry" J. Gerontol., 11, 298-300). These external aggressions
can include: ultraviolet radiation, toxins, atmospheric pollutants,
alimentary oxidants. Ultraviolet radiations skin exposure induces
extensive generation of reactive oxygen species (ROS). These can
react with DNA, proteins, fatty acids and saccharides causing
oxidative damage.
[0007] In the skin, premature aging is observed, occurring in the
areas exposed to ultraviolet radiation, characterized by phenomena
of alteration to the macromolecules (lipidic peroxidation,
carbonylation of proteins), affecting in particular elastin,
collagen or fibronectin.
[0008] One of the important consequences of the accumulation of
oxidative damage is a reduction in the capacity of the cell to
produce ATP (Porteous et al., Eur J Biochem 1998, 257(1):192-201).
Thus, the phenomenon of cellular aging is in relation to the
oxidative damage which the cell undergoes, but also to the process
of energy production necessary for the cell to survive.
[0009] The human sirtuin family comprises 7 proteins, very
conserved throughout evolution, named SIRT1 to SIRT7. "SIRT2
protein" is predominantly located in the cytoplasm and plays roles
in oxidative stress response, inflammation, mitotic progression,
microtubule dynamics, cell migration, longevity. In cytoplasm,
SIRT2 co-localizes with and deacetylates microtubules. During
mitosis, it is translocated to the nucleus where it deacetylates
histone and regulates chromosomal condensation (Serrano L. et al.,
"The tumor suppressor SirT2 regulates cell cycle progression and
genome stability by modulating the mitotic deposition of H4K20
methylation", Genes & Dev. 2013. 27: 639-653). Thereby, SIRT2
plays mitotic checkpoint role: it regulates the mitotic progression
and the mitotic exit (Bosch-Presegue L. and Vaquero A., "Sirtuins
in stress response: guardians of the genome", Oncogene (2014) 33,
3764-3775). Moreover, the stability of spindle assembly checkpoint
protein BubR1 is under control of SIRT2 and a decline in BubR1 over
time has been linked to mammalian aging (North B. J. et al., "SIRT2
induces the checkpoint kinase BubR1 to increase lifespan", The EMBO
Journal, 2014, 33, Issue 13, 1438-1453). SIRT2 can therefore be
associated with increased cell longevity through BubR1 pathway
preservation.
[0010] Deacetylation of Lys668 of BubR1 by SIRT2 inhibits the
ubiquitination of BubR1 and its designation to the proteasome.
Germline mutation that reduce BubR1 abundance cause aneuploidy.
[0011] From the state of the art, numerous cosmetics are known
which in some way contain plant-based raw materials in the form of
oils or extracts. In most cases, the known advantageous effects of
individual plants are used to achieve a corresponding overall
effect.
[0012] The aim of the invention was to develop a novel plant
extract exhibiting activation of the SIRT2 expression. Patent
EP1868632 disclosed synthetic peptides able to activate endogenous
synthesis of sirtuin (SIRT1) protein and have been identified by
inventors to be able to activate SIRT 2. Based on this knowledge,
the inventors searched for botanicals comprising sequence homology
to those identified activating SIRT2 peptides. Sequence similarity
searches to identify homologous sequences were performed using
comprehensive protein sequence data bases like BLAST following
standard procedures (William Pearson, "An Introduction to Sequence
Similarity ("Homology") searching", Curr Protoc Bioinformatics.
June 2013). This search allowed the inventors to select Prunus
persica as a good candidate.
[0013] The peach flower extract according to the invention has
shown an increased expression of sirtuin SIRT2 proteins in the
skin.
[0014] The species name Prunus persica refers to its widespread
cultivation in Persia, whence it was transplanted to Europe. It
belongs to the genus Prunus which includes the cherry and plum, in
the family Rosaceae. Genetic studies suggest peaches originated in
China, where they have been cultivated since the early days of
Chinese culture circa 2000 BC.
[0015] Prunus persica has long been used in Chinese medicine for
treating skin disorder. In Korea, flowers have always been a
favorite source of inspiration for women and men of letters. In
addition to this quality of muse, flowers have been used in the
life of every day, for food decoration or infusions.
[0016] The peach blossom is the symbol of Korea Feminine Beauty.
The infusion with peach blossoms was once very popular among women
of high social rank who knew its beneficial properties for the skin
tone. Peach blossom, popularly consumed as tea, is believed to
promote healthy, young-looking skin. The literature reporting
pharmacological studies related to peach blossom is very
limited.
[0017] Peach flower is known to be very rich in polyphenolic
compounds, such as phenolic acids, and flavonoids. These molecules
can act as scavengers of superoxide anions, singlet oxygen,
hydroxyl radicals, and lipid peroxyl radicals. Many flavonoids such
as quercetin, luteolin and catechins are better antioxidants than
the nutrients vitamin C and beta-carotene (Svobodova et al.,
"Natural phenolics in the prevention of UV-induced skin damage. A
Review" Biomed. Papers 147(2), 137-145 (2003)). The controlled
hydrolysis enables these compounds to be released. The peach leaves
are used as an anthelmintic, and sedative in traditional medicine
(Nadkarni, 1976). Cevallos-Casals et al. reported that peach fruits
rich in phenolic and anthocyanin have good antioxidant and
antimicrobial activities (Cevallos-Casals et al., "Selecting new
peach and plum genotypes rich in phenolic compounds and enhanced
functional properties", Food Chemistry 96 (2006) 273-280). The
pink-colored peach blossom flowers, popularly consumed as tea, are
purgative and are believed to promote healthy, young-looking skin.
The peach blossom is the symbol of Korea Feminine Beauty. The
infusion with peach blossoms was once very popular among women of
high social rank who knew its beneficial properties for the skin
tone.
[0018] Some flavonoids have been identified in peach flower such as
the kaempferol glycoside derivatives multiflora B, trifolin,
afzelin, and astragalin. In addition, a skin protection effect was
demonstrated for multiflora B (Young ha Kim et al., "The extract of
the flowers of Prunus persica, a new cosmetic ingredient, protects
against solar ultraviolet-induced skin damage in vivo", j. Cosmet.
Sci., 53, 27-34 (January/February 2002)).
[0019] Peach flowers which are pink in color have been used to
obtain the extract. Flowers are rich in polyphenolic compounds and
also in flavonoids in particular pink flower are also rich in
particular flavonoids, the anthocyanins that give the pink color to
the flower in comparison to white flower.
[0020] Anthocyanins have been demonstrated to have a broad spectrum
of biological functions and may act as good antioxidant like other
members of flavonoids family
[0021] Antioxidants play an important role as health protecting
factor. Primary sources of naturally occurring antioxidants are
whole grains, fruits, flower and vegetables. Plant antioxidants are
vitamin C, vitamin E, carotenes, phenolic acid, flavonoids. They
have been recognized as having the potential to reduce disease
risk, anti-aging effect. The antioxidant effect of pink peach
blossom extract has been investigated according to the method of
2,2-Diphenyl-1-picrylhydrazyl (DPPH).
[0022] The use of peach extract in a cosmetic composition is known
in prior art. Most extract are total extracts or water-alcohol
extracts. For example, the Japanese patent application,
JP-A-2001302439 has disclosed a cosmetic having inhibitory action
on elastases and collagenases for prevention of skin aging by
including an extract from white Prunus Persia Batsh obtained by
high temperature extracting process. Korean patent, KR-526637 has
disclosed an anti-ultraviolet and cosmetic composition for skin
aging protection including ferulic acid and peach blossom extract
in which the stability of skin including the extract from peach
Blossom of 0.1-20.0 weight % and ferulic acid of 0.1-20.0 weight %
in the active ingredient is improved. Japanese patent application,
JP-A-2016053008 has disclosed a manufacturing method of the peach
extract which has the active oxygen eliminating ability carrying
out partition extraction of the obtained extract with hexane/water
further, and contains sphingoglycolipid (ceramide component).
Korean patent, KR-1429117 has disclosed a composition for skin
whitening containing peach blossom extract obtained by a step of
removing the flower (fuse) from the peach blossom and using only
the calyx, drying the sample, pulverizing the sample, subjecting to
the homogenizer. With the step of extracting in the solvent
selected in the group consisting of water, lower alcohol of the
C3-6 and lower organic acid of the C3-6. The obtained extract is
freeze-dried after it filtering with filter paper. Unlike the prior
art, the present application provides an effective process for
extraction of peach flower extract with high polyphenol content and
free of ceramide.
[0023] In spite of the various anti-aging cosmetic products on the
market for the treatment of skin, there remains a need for
effective topically applied cosmetic compositions that provide
anti-aging or rejuvenating benefits to the skin, hair and/or nails
using natural ingredients as active agent. Unnatural,
chemically-synthesized products may be perceived as being
environmentally or personally unsafe. In contrast, natural products
are perceived as pure, mild, and superior to chemically synthesized
products. Numerous natural based products extracted from plants or
herbs are known to contain antioxidant/free-radical scavenging
agents that can neutralize the effects of free-radical damage.
Additionally, they can contain agents that stimulate the synthesis
and restoration of damaged connective tissue structures in the
dermis and barrier function in the epidermis.
[0024] There remains a need for cosmetic compositions which address
the signs of aging, in particular the appearance of wrinkles,
lines, and sagging. It is therefore an object of the present
invention to provide new compositions and methods for treating,
ameliorating, and/or preventing signs of aged or aging skin. It is
a further object of the invention to improve the overall appearance
of aging or aged skin.
[0025] The foregoing introduction is presented solely to provide a
better understanding of the nature of the problems confronting the
art and should not be construed in any way as an admission as to
prior art nor should the citation of any reference herein be
construed as an admission that such reference constitutes "prior
art" to the instant application.
SUMMARY
[0026] The main aspect of the present invention provides a process
for obtaining an extract containing flowers of plants of the genus
Prunus persica L., comprising the following steps: [0027] (i)
adding water to peach flower to make a mixture, [0028] (ii)
agitating the said mixture by maintaining temperature from room
temperature (RT) to above 70.degree. C. [0029] (iii) filtering the
mixture to remove the solid flower part to obtain the extract.
[0030] In another aspect, the present invention provides a cosmetic
composition comprising peach flower extract obtained by aqueous
extraction, wherein the peach flower extract comprises compounds
having a molecular weight of less than 10 kDa in a physiologically
acceptable medium.
[0031] In yet another aspect, the present invention provides a
method for reducing and/or correcting the signs of aging and
photo-aging of the skin and keratinous appendages, comprising
topically applying to the skin, the mucous membranes and/or
superficial skin appendages a cosmetic composition comprising peach
flower extract obtained by aqueous extraction, wherein the peach
flower extract comprises compounds having a molecular weight of
less than 10 kDa in a physiologically acceptable medium.
[0032] The yet another aspect, the present invention provides
method for modulating the SIRT2 expression, comprising topically
applying to the skin, the mucous membranes and/or superficial skin
appendages a cosmetic composition comprising peach flower extract
obtained by aqueous extraction, wherein the peach flower extract
comprises compounds having a molecular weight of less than 10 kDa
in a physiologically acceptable medium.
BRIEF DESCRIPTION OF THE DRAWINGS
[0033] Further embodiments of the present invention can be
understood with the appended figures.
[0034] FIG. 1 is illustration of quantification of SIRT2 mRNA study
on keratinocytes
[0035] FIG. 2 is illustration of quantification of SIRT2 mRNA study
on fibroblasts
[0036] FIG. 3 is illustration of quantification of BubR1 mRNA study
on keratinocytes
[0037] FIG. 4 is illustration of Senescent associated
beta-galactosidase (SA beta-gal) activity study on fibroblasts
[0038] FIG. 5 is illustration of microscopic observation of BubR1
staining on fibroblasts
[0039] FIG. 6 is illustration of the activity of beta-galactosidase
senescent marker on fibroblasts
[0040] FIG. 7 is illustration of quantification of fibroblasts
through a fluorescent marker system
[0041] FIG. 8 is illustration of the average cell number measured
through absorbance @ 570 nm
DETAILED DESCRIPTION
[0042] Detailed embodiments of the present invention are disclosed
herein; however, it is to be understood that the disclosed
embodiments are merely illustrative of the invention that may be
embodied in various forms. Therefore, specific structural and
functional details disclosed herein are not to be interpreted as
limiting, but merely as a representative basis for teaching one
skilled in the art to variously employ the present invention.
[0043] Whenever a term is identified by reference to a range, the
range will be understood to explicitly disclose every element
thereof. As a non-limiting example, a range of 1-10% will be
understood to include 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, and 10%,
and all values between 1 and 10%.
[0044] Where two or more substituents are referred to as being
"selected from" a group of enumerated alternatives, it is meant
that each substituent can be any element of that group, independent
of the identity of the other substituents.
[0045] As used herein, "% refers % by weight, that is the weight
percent of a component in relation to the total weight of the
composition (i.e., including any carriers, vehicles, solvents,
fillers, or other components added before application to the skin)
unless otherwise provided.
[0046] All terms used herein are intended to have their ordinary
meaning unless otherwise provided. For the purposes of describing
and claiming the present invention, the following terms are
defined:
[0047] "Extract" is understood to be any substance or isolated
preparation extracted from a natural source, regardless of
extraction method or ingredients. The term is used in a broad sense
including, for example, ingredients soluble in water or an organic
solvent extracted from a natural substance using the solvent, or
specific ingredients of a natural substance.
[0048] "Aqueous extract" is understood to be a mixture of compounds
obtained by extraction with water.
[0049] The term "peptide" indicates a sequence of two or several
amino acids linked together by peptide bonds or by modified peptide
bonds; and a polypeptide indicates a peptide of larger size. The
term "peptide" refers to a natural or synthetic peptide of the
invention as described above or at least any natural or synthetic
peptide whose sequence is totally or partly constituted by the
sequence previously described.
[0050] It is understood by "physiologically acceptable" that the
active agent according to the invention, or a composition
containing said agent, is suitable for coming into contact with the
skin or a mucus membrane without provoking a toxicity or
intolerance reaction.
[0051] "Cutaneous signs of aging and photo-aging" refers to all
changes in the external appearance of the skin and skin appendages
due to aging, such as, for example, thinning of the skin, sagging,
loss of hydration and atonia, deep wrinkles and fine lines, loss of
firmness and tone, dermal atrophy or any other internal degradation
of the skin resulting from exposure to ultraviolet radiation, liver
spots and age spots. Liver spots also known as "Solar lentigo",
"Lentigo senilis", "Old age spot", "Senile freckle", are blemishes
on the skin associated with aging and photo-aging due to exposure
to ultraviolet radiation from the sun. They range in color from
light brown to red or black and are located in areas most often
exposed to the sun, particularly the hands, face, shoulders, arms
and forehead, and the scalp if bald.
[0052] "Anti-Aging Benefit" Anti-aging benefits include, but are
not limited to, one or more of: (a) treatment, reduction, and/or
prevention of fine lines or wrinkles, (b) reduction of skin pore
size, (c) improvement in skin thickness, plumpness, and/or
tautness; (d) improvement in skin suppleness and/or softness; (e)
improvement in skin tone, radiance, and/or clarity; (f) improvement
in procollagen and/or collagen production; (g) improvement in skin
texture and/or promotion of retexturization; (h) improvement in
skin barrier repair and/or function; (i) treatment and/or
prevention of skin sagging or atrophy; (j) improvement in
appearance of skin contours; (k) restoration of skin luster and/or
brightness; (l) replenishment of essential nutrients and/or
constituents in the skin; (m) improvement of skin appearance
decreased by menopause; (n) improvement in skin moisturization
and/or hydration; and (o) improvement of skin elasticity and/or
resiliency.
[0053] The species name "Prunus persica" in the present invention
refers to its widespread cultivation in Persia, whence it was
transplanted to Europe. It belongs to the genus Prunus which
includes the cherry and plum, in the family Rosaceae. Genetic
studies suggest peaches originated in China, where they have been
cultivated since the early days of Chinese culture circa 2000 BC.
Prunus persica has long been used in Chinese medicine for treating
skin disorder (Young ha Kim et al., "The extract of the flowers of
Prunus persica, a new cosmetic ingredient, protects against solar
ultraviolet-induced skin damage in vivo", j. Cosmet. Sci., 53,
27-34, January-February 2002).
[0054] As used herein, "skin" refers to all of the covering tissue
constituting the skin, the mucous membranes and the skin
appendages, including hair, eyelashes and eyebrows.
[0055] "Topical application" is understood to be the application or
spreading of a composition containing said peach flower extract, on
the surface of the skin or a mucus membrane.
[0056] What is described herein is a process for obtaining an
extract from dried peach flower, cosmetic composition comprising
the extract, method of reducing and/or correcting the signs of
aging and photo-aging of the skin and keratinous appendages by
topically applying the composition comprising peach flower extract
to the skin and a method for modulating the SIRT2 expression.
[0057] Antioxidants play an important role as health protecting
factors. Scientific evidence suggests that antioxidants reduce the
risk for chronic diseases including cancer and heart disease.
Primary sources of naturally occurring antioxidants are whole
grains, fruits, flower and vegetables. Plant antioxidants are
vitamin C, vitamin E, carotenes, phenolic acid, flavonoids.
[0058] The peach flower extract according to the invention has
shown an increased expression of sirtuin SIRT2 proteins in the
skin. "SIRT proteins" are NAD+ dependent deacetylase and belong to
the Sir2 sirtuin family The deacetylase or
mono-ADP-ribosyltransferase activity of sirtuins enables them to
modulate the acetylation level of some histones. Thus, they often
exert their functions by affecting chromatin, inducing gene
silencing, DNA damage signaling, DNA repair and cell cycle
regulation. Indeed, sirtuins are crucial factors in response to
metabolic, oxidative or genotoxic stresses.
[0059] The peach flower extract according to the invention is known
to be rich in polyphenolic compounds, such as phenolic acids, and
flavonoids. All these water-soluble molecules known for their
antioxidant activity contribute to provide the antioxidant potent
of the peach flower extract according to the invention. Flavonoid
content in the extract was estimated by Aluminum chloride
colorimetric method. The principle involved in aluminium chloride
(AlCl) colorimetric method is that AlCl3 forms acid stable
complexes with the C-4 keto groups and either the C-3 or C-5
hydroxyl group of flavones and flavonols of the extract. The
absorbance of the sample is read on the spectrophotometer at 410
nm. The flavonoid content is determined as rutin equivalent using a
rutin standard curve. Total flavonoids content measured is 200
mg/kg of the extract.
[0060] These molecules can act as scavengers of superoxide anions,
singlet oxygen, hydroxyl radicals, and lipid peroxyl radicals. Many
flavonoids such as quercetin, luteolin and catechins are better
antioxidants than the nutrients vitamin C, vitamin E and
beta-carotene (Svobodova et al., "NATURAL PHENOLICS IN THE
PREVENTION OF UV-INDUCED SKIN DAMAGE. A REVIEW" Biomed. Papers
147(2), 137-145 (2003)). Particularly pink flower are found to be
rich in particular flavonoids, the anthocyanins that give the pink
color to the flower in comparison to white flower.
[0061] In a preferred embodiment, pink flower has been used to
obtain the extract according to the invention.
[0062] Polyphenolic compounds are recognized to be powerful
antioxidant molecules. "Polyphenolic compounds" are compounds found
abundantly in natural plant food sources that have antioxidant
properties. They refer to all the classes of polyphenols, they mean
compounds comprising at least one diphenol aromatic ring, phenol
group may be optionally etherified or esterified. They can also be
called simply "polyphenol". Polyphenols play an important role in
maintaining your health and wellness. Antioxidants as a group help
protect the cells in your body from free radical damage, thereby
controlling the rate at which you age. Antioxidants can be divided
into three major groups: Carotenoids, Allyl sulfides, found in
garlic and onions, Polyphenols (also known as phenolics).
[0063] Polyphenols can be further broken down into four categories:
phenolic acids, flavonoids, lignans, and stilbenes, with additional
subgroupings based on the number of phenol rings they contain, and
on the basis of structural elements that bind these rings to one
another.
[0064] Phenolic acid is a type of phytochemical called polyphenol,
found in a variety of plant-based foods; the seeds and skins of
fruits and the leaves of vegetables contain the highest
concentrations. Phenolic acids are readily absorbed through the
walls of the intestinal tract, and they may be beneficial to health
because they work as antioxidants that prevent cellular damage due
to free-radical oxidation reactions. There are many different
phenolic acids found in nature, and they can be divided into two
categories: benzoic acid derivatives, such as gallic acid; and
cinnamic acid derivatives, including caffeic acid and ferulic acid.
The cinnamic acids are more common than the benzoic acids.
[0065] Flavonoids are the largest family of polyphenolic compounds;
they have structure consisting of 2 aromatic rings (A and B) that
are bound together by 3 carbon atoms that form an oxygenated
heterocycle (ring C). Flavonoids are further divided into 6
subclasses as a function of the type of heterocycle involved:
flavonols, flavones, isoflavones, flavanones, anthocyanidins, and
flavanols (catechins and proanthocyanidins). Plants produce
flavonoids as a protection against parasites, oxidative injury and
harsh climatic conditions.
[0066] There are three major classes of plant flavonoids
(anthocyanins, proanthocyanins, and flavonols), synthesized via the
branched flavonoid biosynthetic pathway. Anthocyanins, the most
important flavonoid class, are the main pigments in flowers and
fruits; these pigments are vital for insect attraction, necessary
for pollination and seed dispersal (Winkel-Shirley, "Flavonoid
Biosynthesis. A Colorful Model for Genetics, Biochemistry, Cell
Biology, and Biotechnology", Plant Physiol. Vol. 126, 2001). The
basic anthocyanins consist mainly of pelargonidin, cyanidin, and
delphinine; various modifications of these compounds, such as
glycosylation, acylation, and methylation may occur, contributing
to diversity in flower color (Morata et al., "Formation of the
highly stable pyranoanthocyanins (vitamins A and B) in red wines by
the addition of pyruvic acid and acetaldehyde", Food Chemistry 100
(2007) 1144-1152). Anthocyanins have been also demonstrated to have
a broad spectrum of biological functions and may act as good
antioxidant like other members of flavonoids family
[0067] Flower pigmentation involves a complex multi-enzymatic
biosynthetic pathway, in which several enzymatic reactions result
in the production and accumulation of flavonoid compounds.
Flavonoids play important roles in many biological processes, such
as UV protection (Li et al., "Arabidopsis Flavonoid Mutants Are
Hypersensitive to UV-6 Irradiation", The Plant Cell, Vol. 5,
171-179, February 1993), pathogen defense (Treutter, "Significance
of Flavonoids in Plant Resistance and Enhancement of Their
Biosynthesis", Plant Biology 7 (2005) 581-591), and pollen
viability (Taylor, L P and Jorgensen R (1992). "Conditional male
fertility in chalcone synthase-deficient petunia". J. Hered. 83:
11-17.). Peach flower contains particular flavonoids, four
kaempferol glycoside derivatives (multiflorin B, trifolin, afzelin,
and astragalin) and a skin protection effect was demonstrated for
multiflorin B (Young ha Kim et al., "The extract of the flowers of
Prunus persica, a new cosmetic ingredient, protects against solar
ultraviolet-induced skin damage in vivo", j. Cosmet. Sci., 53,
27-34 (January/February 2002))
[0068] Among the "flavonoids" used in the invention, may be
mentioned in particular taxifolin, catechin, epicatechin,
eriodictyol, naringenin, rutin, troxerutin, chrysin, tangeretin,
luteolin, epigallocatechin gallate and epigallocatechin, quercetin,
fisetin, kaempferol, galangin, gallocatechin, epicatechin
gallate.
[0069] (Hassani et al., (2015), "Analysis of biochemical compounds
and differentially expressed genes of the anthocyanin biosynthetic
pathway in variegated peach flower", Genetic Mol Res.
14(4):13425-36) provides a comparative study by LC-MS analysis of
the compounds produced in flower petals of different colors (white
and pink) from a marked, variegated peach tree. Several
glycosylated derivatives of cyanidin (cyanidin-3-glucoside,
cyanidin-3-(6''-malonyl glucoside), cyanidin-3-(6''-ethylmalonyl
glucoside)) also pelargonidin (pelargonidin-3-glucoside,
Pelargonidin-3-(6''-ethylmalonyl glucoside) and peonidin
(peonidin-3-glucoside and Peonidin-3-(6''-ethylmalonyl glucoside))
were detected in pink petal however; none of these compounds were
detected in white extracts. These compounds in particular cyanidin
have been described for their antioxidant and anti-inflammatory
effect as described in the work of Jung et al published in 2014.
They have been recognized as having the potential to reduce disease
risk, anti-aging effect. The antioxidant effect of pink peach
blossom extract has been investigated according to the method of
2,2-Diphenyl-1-picrylhydrazyl (DPPH).
[0070] The antioxidant effect of pink peach blossom extract
according to the invention has been investigated according to the
method of DPPH. DPPH assay is a rapid, simple method to measure
antioxidant capacity involving the use of the free radical,
2,2-Diphenyl-1-picrylhydrazyl (DPPH) which is widely used to test
the ability of compounds to act as free radical scavengers or
hydrogen donors and to evaluate antioxidant activity.
[0071] The DPPH assay method is based on the reduction of DPPH, a
stable free radical. The free radical DPPH with an odd electron
gives a maximum absorption at 515 nm (purple colour). When a
solution of DPPH is mixed with a substance that can donate a
hydrogen atom, then this gives rise to the reduced form
(Diphenylpicrylhydrazine; non radical) with the loss of this violet
colour to a residual pale yellow colour from the picryl group still
present (Prieto P, Pineda M, and Aguilar M (1999),
"Spectrophotometric quantitation of antioxidant capacity through
the formation of a phosphomolybdenum complex: Specific application
to the determination of vitamin E", Anal Biochem 269, 337-341).
[0072] One of the advantages of the present invention is that the
pink peach flowers are highly enriched in different compounds of
interest compared to leaves, fruits, roots or any other parts of
the plant.
[0073] The present invention provides a peach flower extract
obtained from Prunus persica species and more preferably the Peach
flower extract is obtained from the whole flower.
[0074] According to the invention "whole flower" or "peach flower",
comprises both petals and sepals. Flowers maybe either fresh or
dried. Preferably, the flowers are dried by natural shadow air
drying or at 45.degree. C. for 48 hours or at 15.degree. C. for 72
hours in a hot air oven.
[0075] The peach flower extract according to the invention can be
obtained by aqueous extraction. A large number of compounds found
in peach extract are likely to have biologically activity are water
soluble.
[0076] In a preferred embodiment, the present invention provides a
process for obtaining an extract from peach flower (Prunus persica
L.), said process comprising: [0077] (i) adding water to peach
flower to make a mixture, [0078] (ii) agitating the said mixture
for 2 hours by maintaining temperature from RT to below 70.degree.
C. [0079] (iii) filtering the mixture to remove the solid flower
part to obtain the extract. [0080] (iv) pasteurizing the mixture
overnight at a temperature below 70.degree. C.
[0081] Flowers may be either fresh or dried. In a preferred
embodiment, flowers are dried and water is added to the dried
flowers to make a mixture. Any of the well-known methods of drying,
such as natural shadow or oven drying can be performed. In a most
preferred embodiment flowers are dried in an oven during 48 h to 72
hours at a temperature of 15.degree. C. to 45.degree. C.
[0082] In a preferred embodiment the peach flower material is
macerated in water. The solution is subjected to short time gentle
maceration for 2 hours at a temperature between RT to less than
70.degree. C. Most preferably the temperature is maintained between
RT to 50.degree. C. to preserve the integrity of molecules of
interest such as flavonoids and phenolic acids and so their ability
to act as antioxidant.
[0083] The selection of the extraction temperature depends on the
desired type of compounds to be extracted, the structural
characteristics of the botanical source (flowers, fruits, stems,
seeds, leaves, root), the quality and yield required for the
extract, and the economic feasibility for scaling up the process.
The extraction of the phenolic compounds from the plant material is
influenced by the extraction temperature and time, which reflects
the conflicting actions of solubilization and analyte degradation
by oxidation. However, many phenolic compounds are easily
hydrolyzed and oxidized. Long extraction times and high temperature
increase the chance of oxidation of phenolics which decrease the
yield of phenolics in the extracts. The analysis was done at
different temperatures and showed that high temperatures degrade
these types of molecules. The extract has complex structures and
with heat they are destructured and lose their potential biological
activity. High temperatures have been shown to cause rapid
anthocyanin degradation. Indeed long time extraction and high
temperature (70.degree. C. and above) increase the chance of
oxidation which decrease yield of polyphenolic compounds in the
extract, also a pH around 4-5 is suitable for stability of
polyphenolic compounds. Therefore, it is of critical importance to
select efficient extraction temperature to obtain and maintain the
stability of phenolic compounds.
[0084] The water extraction can be carried out at room temperature
or with water heated at no more than 70.degree. C., while being
agitated. Preferably, the extraction is carried out by maceration
in water heated at 50.degree. C. for 2 hours. The raw solution is
then subjected to grid filtering to remove insoluble material.
After grid filtering, the aqueous or liquid fraction is collected.
To remove smaller residues of the aqueous extract, a filtration by
any process well known by someone skilled in the art may be carried
out.
[0085] In a preferred embodiment, the purification process begins
by successive filtrations using filters with decreasing porosity
from 50-20 .mu.m until 0.5-0.2 .mu.m to get an extract. In another
preferred embodiment, the extract obtained is composed of protein
fragments and peptides with a molecular weight of less than 10 kDa,
as demonstrated by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). The obtained extract is a clear and
brilliant solution.
[0086] In another preferred embodiment, the extract is then diluted
at a concentration between 8 g/Kg and 13 g/Kg, preferably at 11
g/Kg of dry matter with solvents, such as water, glycerol, ethanol,
propanediol, butylene glycol, dipropylene glycol, ethoxylated or
propoxylated glycols, cyclic polyols or any mixture of these
solvents for example 30% glycerol and 0.85% of phenoxyethanol by
maintaining the pH between 4.0 to 4.5.
[0087] Then, the diluted active agent is sterilized by sterile
filtration, Then the solution is heated overnight at 65.degree. C.
to perform low-temperature pasteurization.
[0088] Furthermore, the diluted active agent according to the
invention can be qualitatively and quantitatively analyzed. The
characteristics are the following: [0089] Proteins: 3-6 g/kg,
[0090] Sugars: 3-6 g/kg, [0091] Amino acids: 0.5-1.5 g/kg, [0092]
Phenolic compounds: 0.5-1.5 g/kg, [0093] Flavonoids: 0.1-0.4 g/kg.
[0094] The extract does not contain any ceramide or
sphingolipid.
[0095] Protein contents of the peach flower extract have been
determined by Lowry protein assay (Lowry O H, Rosebrough N J, Farr
A L, Randall R J (1951). "Protein measurement with the Folin phenol
reagent", J. Biol. Chem. 193 (1): 265-75) which has been used to
quantify total protein content of the extract. The Lowry assay is a
biochemical assay for determining the total level of protein in a
solution. The Lowry method is based on the reaction of Cu+,
produced by the oxidation of peptide bonds, with Folin-Ciocalteu
reagent. The absorbance of the sample is read on the
spectrophotometer at 550 nm. The protein content is determined
using a BSA standard curve.
[0096] Aminoacid content of the peach flower extract have been
determined starting from a protocol published by Moore et al.
(Moore et al, "Photometric ninhydrin method for use in the
chromatography of amino acids", Journal of Biological Chemistry
1948 Vol. 176 pp. 367-388). The free amino acid content of the
extract was assessed by the formation of a colored complex,
following the rupture of the amine and carboxylic functions by the
reagent ninhydrin. The absorbance of the complex is read on the
spectrophotometer at 570 nm. The total amino acids content is
determined using a standard curve of amino acids pool.
[0097] Total sugars content on the peach flower extract was
determined colorimetrically via an adaptation of the assay
described by (Dubois et al. ("Colorimetric Method for Determination
of Sugars and Related Substances", Anal. Chem., 1956, 28 (3),
350-356). This analysis consists in concentrated sulfuric acid
reacting with phenol to form a colored complex. The absorbance of
the complex is read on the spectrophotometer at 490 nm. The sugar
content is determined using a glucose standard curve.
[0098] Polyphenols content of the peach flower extract was
determined using the Folin-Ciocalteu assay (Singleton et al.
(1999). "Analysis of total phenols and other oxidation substrates
and antioxidants by means of Folin-Ciocalteu reagent", 299: 152).
Polyphenols compounds in the sample react with the Folin-Ciocalteu
reagent, the oxidation of the reagent give a blue color. The
absorbance of the sample is read on the spectrophotometer at 760
nm. The content was expressed as gallic acid equivalents using a
gallic acid standard curve.
[0099] SDS PAGE electrophoresis was performed to assess molecular
weight of proteins of the extract. The peach flower extract is
heated to 70.degree. C. for 10 minutes in reductive denaturing
conditions in a denaturing sample buffer. An antioxidant solution
is added to the inner chamber (cathode) so that the reduced
proteins do not re-oxidize during electrophoresis. Protein
migration is carried out using the MES running buffer with standard
Novex.RTM. Sharp as a marker for molecular weight. Protein staining
is carried out using silver staining.
[0100] The advantage of the extract according to the invention is
that small compounds are more stable and reproducible without
having an allergenic effect.
[0101] In another aspect, the present application provides a
cosmetic composition comprising peach flower extract obtained by
aqueous extraction, wherein the peach flower extract comprises
compounds having a molecular weight of less than 10 kDa and a
physiologically acceptable medium.
[0102] In another preferred embodiment, the peach flower extract is
present in a concentration range from about 0.0001% to about 20% by
weight, preferably 0.0005% to about 5% by weight of the total
weight of the composition.
[0103] In another embodiment, the peach flower extract is used for
cosmetic applications, more preferably for topical
applications.
[0104] In another preferred embodiment, the present invention
provides oral, parenteral or topical formulations adapted by the
person skilled in the art, in particular for cosmetic or
dermatological compositions. The compositions according to the
invention are advantageously designed to be administered topically.
These compositions must therefore contain a physiologically
acceptable medium, i.e. compatible with the skin and epithelial
appendages, and cover all cosmetic or dermatological forms.
[0105] Further, the present compositions preferably are in the form
of an aqueous, hydroalcoholic or oily solution; oil-in-water
emulsion, water-in-oil emulsion or multiple emulsions; creams,
suspensions, powders adapted for application on the skin, mucus
membranes, lips and/or epithelial appendages. These compositions
can also be more or less fluid and have the appearance of a cream,
a lotion, milk, a serum, pomade, a gel, a paste or a mousse. They
can also exist in solid form, as a stick, or can be applied to the
skin as an aerosol. They can also be used as a skincare product
and/or as a makeup product.
[0106] In another embodiment, the composition comprises
conventionally used additive envisaged in the scope of application
as well as necessary additives for their formulation, such as
co-solvents (ethanol, glycerol, benzyl alcohol, damper . . . ),
thickeners, thinners, emulsifiers, antioxidants, colorants, solar
filters, pigments, fillers, preservatives, perfumes, odor
absorbers, essential oils, oligo elements, essential fatty acids,
surfactants, film-forming polymers, chemical filters or minerals,
moisturizing agents or thermal waters etc. Water-soluble,
preferably natural, polymers, such as polysaccharides or
polypeptides, cellulose derivatives of the type methylcellulose or
hydroxypropylcellulose, or even synthetic polymers, poloxamers,
carbomers, siloxanes, PVA or PVP, and in particular polymers sold
by the company Ashland, can be cited, for example.
[0107] It is well understood that the active agent according to the
invention can be used on its own or in conjunction with other
active ingredients.
[0108] Furthermore, the compositions which can be used according to
the invention advantageously contain at least one other active
agent. The following types of ingredients can be cited, in a
non-limiting manner: other peptide active agents, vegetable
extracts, healing agents, anti-aging agents, anti-wrinkle agents,
soothing agents, anti-free radicals, anti-ultraviolet radiation
agents, agents for stimulating dermal macromolecular synthesis or
energetic metabolism, moisturizing agents, antibacterial agents,
antifungal agents, anti-inflammatories, anesthetics, agents
modulating cutaneous differentiation, cutaneous pigmentation or
depigmentation, and agents for stimulating nail and hair
growth.
[0109] It is preferable for an anti-free radical or antioxidant
agent, or an agent stimulating dermal macromolecular synthesis or
energetic metabolism, to be used.
[0110] In a more specific embodiment, the composition according to
the invention will comprise: [0111] Sunscreens, ultraviolet and
Infra-red screens [0112] Anti-free radical agents, [0113] DHEA
(dehydroepiandrosterone), [0114] At least one cytochrome
co-activating compound, and/or; [0115] One (or more)
aquaporin-activating compound and/or; [0116] One (or more)
sirtuin-activating compound and/or; [0117] One (or more) compound
that increases cell adhesion and/or; [0118] One (or more) compound
that increases the production of matrix proteins of the collagen or
laminin type, etc.; [0119] One (or more) HSP protein-modulating
compound; [0120] One (or more) compound that increases cell energy;
[0121] One (or more) pigmentation-modulating compound such as a
yeast, amaranth, linseed, bean, cacao, corn, soy, sunflower,
rapeseed or pea peptide extract; [0122] One (or more) compound
improving the skin barrier function; [0123] One (or more)
mitochondria-protecting compound. [0124] Vitamin A and notably
retinoic acid, retinol, retinol proprionate, retinol palmitate,
[0125] Vitamin B3 and notably niacinamide, niconitate of
tocopherol, [0126] Vitamin B5, vitamin B6, vitamin B12, panthenol,
[0127] Vitamin C, and notably ascorbic acid, ascorbyl glucoside,
ascorbyl tetrapalmitate, magnesium and sodium ascorbyl phosphate,
[0128] Vitamins E, F, H, K, PP, and coenzyme Q10, Metalloproteinase
inhibitor, activator of Tissue Inhibitor Metalloproteinase (TIMP),
[0129] Aminoacids and notably arginine, ornithine, hydroxyproline,
hydroxyproline dipalmitate, palmitoylglycine, hydroxylysine,
methionine and its derivatives, N-acylated aminoacids, [0130]
Natural or synthetic peptides, including , di-, tri-, tetra-,
penta- and hexapeptides and their lipophilic derivatives, isomers
and complex with other molecules such as metallic ion (i.e. copper,
zinc, manganese, magnesium, and others), peptides sold under
commercial names MATRIXYL.RTM., ARGIRELINE.RTM., CHRONOGEN.TM.,
LAMINIXYL IS.TM., PEPTIDE Q10.TM., COLLAXYL.TM. (patent FR2827170,
ASHLAND.RTM.), PEPTIDE VINCI 01.TM. (patent FK2837098,
ASHLAND.RTM.), PEPTIDE VINCI 02.TM. (patent FR2841781,
ASHLAND.RTM.), ATPeptide.TM. (patent FR2846883, ASHLAND.RTM.) or
synthetic peptide of sequence Arg-Gly-Ser-NH2, sold under
commercial name of ATPeptide.TM. by ASHLAND.RTM.; [0131] Extract of
Artemia salina, sold under commercial name of GP4G.TM. (FR2817748,
ASHLAND.RTM.); [0132] Botanical peptide extracts such as flaxseed
extract (Lipigenine.TM., patent FR2956818, ASHLAND.RTM.), soya
extract, einkorn, grapevine, rapeseed, rice, corn or pea; [0133]
Yeast extracts, such as Dynagen.TM., (patent FR2951946,
ASHLAND.RTM.) or Actopontine.TM. (patent FR2944526, ASHLAND.RTM.);
dehydroacetic acid (DHA), [0134] Natural or synthetic
phystosterols, [0135] alpha- and beta-hydroxyacids, silanols,
[0136] Sugar amines, glucosamine, D-glucosamine,
N-acetyl-glucosamine, N-acetyl-D-glucosamine, mannosamine, N-acetyl
mannosamine, galactosamine, N-acetyl galactosamine, [0137]
Polyphenols, isoflavones, flavonoids, such as grape extract, pine
extract, olive extract, [0138] Lipids such as ceramides or
phospholipids, [0139] Animal oils such as squalenes or squalanes,
[0140] Vegetal oils, such as almond oil, coconut oil, castor oil,
jojoba oil, olive oil, rapeseed oil, peanut oil, sunflower oil,
wheat germ oil, corn germ oil, soybean oil, cotton oil, alfalfa
oil, poppy oil, pumpkin seed oil, evening primrose oil, millet oil,
barley oil, rye oil, safflower oil, passion oil, hazelnut oil, palm
oil, apricot kernel oil, avocado oil, calendula oil, ethoxylated
vegetable oils, or shea butter, the above mentioned compounds can
be natural, such as peptide hydrolysates of plants, or also
synthetic, such as peptide compounds.
[0141] In yet another embodiment, the present application provides
a cosmetic method for reducing and/or correcting the signs of aging
and photo-aging of the skin and keratinous appendages, comprising
topically applying to the skin, the mucous membranes and/or skin
appendages a composition comprising a peach flower extract
according to the application.
[0142] It is clear that the invention is designed for mammals in
general, and more specifically for human beings. The inventors have
indeed identified biological activities which are useful to reduce
and/or correct the cutaneous signs of aging and photo-aging of the
skin and keratinous appendages, and to protect the skin against
aggressions due to ultraviolet radiation.
[0143] "Cutaneous signs of aging and photo-aging" refers to all
changes in the external appearance of the skin and skin appendages
due to aging, such as, for example, thinning of the skin, sagging,
loss of hydration and atonia, deep wrinkles and fine lines, loss of
firmness and tone, dermal atrophy or any other internal degradation
of the skin resulting from exposure to ultraviolet radiation, liver
spots and age spots.
[0144] In yet another aspect, the present application provides a
cosmetic method to protect the skin against aggressions due to
ultraviolet radiation, wherein a cosmetic composition comprising a
peach flower extract according to the invention is applied
topically on the skin which is to be treated.
[0145] In yet another embodiment, the present application provides
a cosmetic method to modulate the SIRT2 expression in skin cells,
wherein a cosmetic composition comprising a peach flower extract
according to the invention is applied topically on the skin which
is to be treated.
[0146] The embodiments which are specific to this cosmetic method
also result from the above description.
[0147] Further advantages and characteristics of the invention can
be seen in greater detail by reading the illustrative, non-limiting
examples provided.
EXAMPLE 1: PREPARATION OF A PEACH FLOWER EXTRACT (PRUNUS PERSICA
L.)
[0148] The pink peach flowers were obtained from Damyang, South
Jeolla, South Korea. The flowers were dried by natural shadow air
drying or at 45.degree. C. for 48 hours or at 15.degree. C. for 72
hours in a hot air oven. 50 g of dry peach flower (Prunus persica)
were placed in 1 liter of distillated water. The solution was
heated 2 hours at a temperature of 50.degree. C. Then a filter grid
was used to separate solid flower residue from the liquid part--The
purification process begins by successive filtrations using filters
of decreasing porosity (from 0.5 to 0.2 .mu.m) so as to obtain a
clear, bright solution. The filtrate was then diluted to obtain an
extract having between 10-12 g/Kg dry matter with 30% glycerol and
0.85% phenoxyethanol. The pH of the solution was adjusted between 4
and 5, to increase the stability of the extract. After
clarification and dilution, the filtrate was then filter-sterilized
with 0.2 .mu.m filter porosity under sterile condition. Then the
solution was heated overnight at 65.degree. C. to perform
low-temperature pasteurization. Peach flower extract was analyzed
using standard procedure. The characteristics of the peach flower
extract obtained are the following: dry matter: 11 g/kg--proteins:
4.5 g/kg--sugars: 4.5 g/kg--amino acids: 0.7 g/kg and polyphenolic
compounds: 1.1 g/Kg. Methods used in spectrophotometry assay to
determine the amount of different compounds in the peach flower
extract: Proteins content of the peach flower extract have been
determined by Lowry protein assay (Lowry et al, 1951) to quantify
total protein content of the extract. The Lowry assay is a
biochemical assay for determining the total level of protein in a
solution. The Lowry method is based on the reaction of Cu+,
produced by the oxidation of peptide bonds, with Folin-Ciocalteu
reagent. The absorbance of the sample is read on the
spectrophotometer at 550 nm. The protein content was determined
using a BSA standard curve. Aminoacid content of the extract have
been determined starting from a protocol published by Moore et al
(1948), the free amino acid content of the extract was assessed by
the formation of a colored complex, following the rupture of the
amine and carboxylic functions by the reagent ninhydrin. The
absorbance of the complex is read on the spectrophotometer at 570
nm. The total amino acids content was determined using a standard
curve of amino acids pool.
[0149] Total sugar content on the extract was determined
colorimetrically via an adaptation of the assay described by Dubois
et al (1956) (Dubois et al, "Colorimetric Method for Determination
of Sugars and Related Substances", Anal. Chem., 1956, 28 (3),
350-356). This analysis consists in the dissolution of the raw
material in concentrated sulfuric acid and then reacting with
phenol to form a colored complex. The absorbance of the complex is
read on the spectrophotometer at 490 nm. The sugar content is
determined using a glucose standard curve.
[0150] Polyphenol content of the peach flower extract was
determined using the Folin-Ciocalteu assay (Singleton et al.,
"Analysis of total phenols and other oxidation substrates and
antioxidants by means of Folin-Ciocalteu reagent", 1999, 299: 152).
Polyphenol compounds in the sample react with the Folin-Ciocalteu
reagent, the oxidation of the reagent gives a blue color. The
absorbance of the sample is read on the spectrophotometer at 760
nm. The content was expressed as gallic acid equivalents using a
gallic acid standard curve
[0151] SDS PAGE electrophoresis was performed to assess molecular
weight of proteins of the extract. The peach flower extract is
heated to 70.degree. C. for 10 minutes in reductive denaturing
conditions in a denaturing sample buffer. An Antioxidant solution
is added to the inner chamber (cathode) so that the reduced
proteins do not re-oxidize during electrophoresis. Protein
migration is carried out using the MES running buffer with standard
Novex.RTM. Sharp as a marker for molecular weight. Protein staining
is carried out using silver staining.
[0152] The extract obtained is composed of peptides with a
molecular weight of less than 10 kDa.
HPLC Protocol
[0153] Chromatographic analysis of phenolic acids: All samples were
separated on a Column Uptisphere CS evolution 100 mm.times.4.6
mm.times.2.6 .mu.m (ref Interchim: UE2.6AQ-100/046) by an Agilent
1260 HPLC system (Agilent Technologies, Calif., USA). The flow rate
was 0.8 ml min-1. The mobile phase consisted of 0.1% TFA
(Trifluoroacetic acids) in aqueous solution (A) and methanol (B).
The elution was facilitated by gradient program as follows (see
table):
TABLE-US-00001 TABLE 1 Gradient of Elution Eau/TFA a Time (min)
0.1% (%) Methanol (%) 0 100 0 7 82 18 30 78 22 35 5 95 40 5 95 42
95 5
The column temperature was maintained at 25.degree. C. The
injection volume was 20 .mu.L and the detection wavelength was set
at 280 nm using a UV detector Amino acids standards were purchased
from Sigma-Aldrich. Identification of amino acids was performed by
comparison of retention times and UV spectral peaks of the sample
with authentic standard. Quantitative estimation of phenolic acids
was done based on the peak area of the sample concentrations and
standard.
[0154] Chromatographic analysis of amino acids: All samples were
separated on a Column Uptisphere Strategy C18-2 5 .mu.m
(250.times.4.6 mm) US5C182-250/046 (ref Interchim: UE2.6AQ-100/046)
by an Agilent 1260 HPLC system (Agilent Technologies, Calif., USA).
The flow rate was 1 ml/min. The mobile phase consisted of
phosphoric acid (H3PO4) 0.1% solution (A) and acetonitrile (B). The
elution was facilitated by gradient program as follows (see
table):
TABLE-US-00002 TABLE 2 Gradient of Elution Acetonitrile Time (min)
H3PO4(0.1%) (ACN) (%) 0 87 13 20 54 46 22 54 46 27 60 40 30 60 40
35 5 95 37 87 13
The column temperature was maintained at 25.degree. C. The
injection volume was 5 .mu.L and the detection wavelength was set
at 254 nm using a UV detector Amino acids standards were purchased
from Sigma-Aldrich. Standard and sample before injection are
derivatized with phenylisothiocyanate (PITC). Identification of
amino acids was performed by comparison of retention times and UV
spectral peaks of the sample with authentic standard. Quantitative
estimation of amino acids was done based on the peak area of the
sample concentrations and standard.
HPLC Analysis
TABLE-US-00003 [0155] TABLE 3 HPLC analysis Process Total amount of
Chlorogenic acid (T.degree. C.) Phenolic acid (mg/Kg) content
(mg/kg) RT 534 422 50.degree. C. 478 342 70.degree. C. 266 220
TABLE-US-00004 TABLE 4 HPLC analysis - Results at a temperature of
50.degree. C. & 70.degree. C. Dry Amino Phenolic Catechin
Process matter acids (mg/ acid (mg/ content (mg/ T.degree. C.
(g/kg) Kg) -HPLC Kg) - HPLC Kg) - HPLC 50.degree. C. 15.3 1432 494
156 70.degree. C. 15.3 1304 214 22
[0156] It was observed that the catechin content is very low at
70.degree. C. compared to 50.degree. C., Catechin belongs to the
group of flavonols, part of the chemical family of flavonoids that
is a type of natural phenol and is a potent antioxidant molecule.
The decreasing content of this molecule with rising temperature may
explain the fact that the extract obtained 70 .degree. C. are less
antioxidant than the extract obtained at a lower temperature.
[0157] As a conclusion it has been shown that an extraction
performed at 50.degree. C. preserves specifically catechin
molecules.
EXAMPLE 2: SIRT2 mRNA STUDY ON KERATINOCYTES AND FIBROBLASTS
TREATED WITH PEACH FLOWER EXTRACT OF EXAMPLE 1, BY qPCR
[0158] The object of this study is to determine the influence of
peach flower extract of example 1 on Sirtuin 2 mRNA level in human
cells, by qPCR.
[0159] Protocol: Treatment solution of synthetic SIRT2 peptide was
prepared by diluting a stock solution of 100 ppm ("ppm" refers to
parts per million) in the culture medium of the cells at 0.5%
vol/vol.
[0160] Treatment solution of peach flower extract were prepared by
diluting peach flower extract of example 1 at 0.1% vol/vol, or a
0.5% vol/vol, in the culture medium of the cells.
[0161] Normal human keratinocytes or normal human fibroblasts were
treated twice with a solution of 0.5% vol/vol, of a synthetic SIRT2
activating peptide or with 0.1% vol/vol or 0.5% vol/vol of peach
flower extract of example 1 for 24 hours. Total RNA was first
extracted using the RNeasy Mini Kit (74106, Qiagen); then, total
RNA was reverse-transcribed with the High Capacity cDNA Reverse
Transcription Kit (4374966, Life technologies). Finally, real-time
PCR was performed on a thermocycler (Applied Biosystems) with
TaqMan Gene Expression Master Mix (4369514, Life technologies) and
TaqMan Gene Expression Assays (Hs00247263_m1, Life technologies)
which were composed of two primers and one probe specific of the
sequence. 18S TaqMan Gene Expression Assay (Hs99999901_s1, Life
technologies) was used as endogenous control and the comparative Ct
method was used for relative quantification of target
expressions.
[0162] Results: The SIRT2 mRNA level was increased after the
treatment with the peach flower extract both on keratinocytes and
fibroblasts, compared to untreated cells. The level was high and
significantly (Student's t test) increased by 64% and 56% after
0.1% treatment and by 88% and 91% after 0.5% treatment on
keratinocytes and fibroblasts respectively.
[0163] Conclusion: The level of SIRT2 mRNA was quantitatively
increased in cells treated with the peach flower extract for 24
hours. Results are illustrated in FIG. 1 & FIG. 2
TABLE-US-00005 TABLE 5 SIRT2 mRNA Expression on Keratinocytes &
Fibroblasts after treatment with peach flower extract Synthetic
SIRT2 activating Peach flower Peach flower Method peptide extract
(0.1%) extract (0.5%) Keratinocytes qPCR +12%*** +64%*** +88%***
Fibroblasts qPCR +27%*** +56%*** +91%***
EXAMPLE 3: SIRT2 PROTEIN EXPRESSION STUDY ON KERATINOCYTES TREATED
WITH PEACH FLOWER EXTRACT OF EXAMPLE 1
[0164] The object of this study is to determine the influence of
peach flower extract of example 1 on Sirtuin 2 protein expression
on cells. To do this, specific labeling by immunofluorescence was
carried out on normal human keratinocytes.
[0165] Protocol: Treatment solution of peach flower extract were
prepared as in example 2.
[0166] Normal human keratinocytes were treated twice with a
solution of 0.5% of a synthetic SIRT2 activating peptide or with
0.1% vol/vol or 0.5% vol/vol of peach flower extract of example 1
for 24 hours. For immunolabelling by anti-SIRT2 antibody, the cells
were washed and fixed with paraformaldehyde at 3.7% for 10 minutes.
The cells were then incubated in the presence of a specific
anti-SIRT2 antibody (Abcam, ref. ab19388, rabbit polyclonal), and
then a secondary suitable antibody, coupled with a fluorescent dye.
After mounting in a particular medium, the slides were observed by
epifluorescence microscope (Nikon Eclipse 80i microscope).
[0167] Results: Microscopic observation showed an increased SIRT2
staining on keratinocytes, after the treatment with the peach
flower extract.
[0168] Conclusion: The expression of SIRT2 protein was visually
increased in cells treated with the peach flower extract for 24
hours.
EXAMPLE 4: SIRT2 PROTEIN EXPRESSION STUDY ON EX VIVO HUMAN SKIN
BIOPSIES TREATED WITH PEACH FLOWER EXTRACT OF EXAMPLE 1
[0169] The object of this study is to determine the influence of
peach flower extract of example 1 on Sirtuin 2 protein expression
on skin. To do this, specific labeling by immunofluorescence was
carried out on normal human skin biopsies.
[0170] Protocol: Treatment solution of peach flower extract were
prepared as in example 2.
[0171] Normal human skin biopsies were treated twice with a
solution of 0.1% vol/vol or 0.5% vol/vol of peach flower extract of
example 1 for 24 hours. For immunolabelling by anti-SIRT2 antibody,
tissues were fixed and embedded in paraffin. Embedded skin biopsies
were then cut and sections were deparaffinized and rehydrated.
Then, an unmasking protocol was performed before applying a
specific anti-SIRT2 antibody (Abcam, ref. ab19388, rabbit
polyclonal), and then a secondary suitable antibody, coupled with a
fluorescent dye. After mounting in a particular medium, the slides
were observed by epifluorescence microscope (Nikon Eclipse 80i
microscope).
[0172] Results: Microscopic observation of SIRT2 immunofluorescent
staining on skin sections showed an increase of SIRT2 expression
after the treatment with the peach flower extract, in a dose
dependent manner, as shown in table 5. The increase was in a
dose-dependent manner and higher in comparison to the treatment
with the synthetic peptide.
[0173] Conclusion: The expression of SIRT2 protein was visually
increased in skin biopsies treated with the peach flower extract
for 24 hours.
EXAMPLE 5: BubR1 mRNA STUDY ON KERATINOCYTES TREATED WITH PEACH
FLOWER EXTRACT OF EXAMPLE 1, BY qPCR
[0174] As BubR1 protein is under control of SIRT2 and is linked to
mammalian aging, the object of this study is to determine the
influence of peach flower extract of example 1 on BubR1 mRNA level
in human cells, by qPCR.
[0175] Protocol: Treatment solution of peach flower extract were
prepared as in example 2.
[0176] Normal human keratinocytes were treated twice per day with a
solution of 0.1% vol/vol or 0.5% vol/vol of peach flower extract of
example 1 for 24 hours. Total RNA was first extracted using the
RNeasy Mini Kit (74106, Qiagen); then, total RNA was
reverse-transcribed with the High Capacity cDNA Reverse
Transcription Kit (4374966, Life technologies). Finally, real-time
PCR was performed on a thermocycler (Applied Biosystems) with
TaqMan Gene Expression Master Mix (4369514, Life technologies) and
TaqMan Gene Expression Assays (Hs01084828_m1, Life technologies)
which were composed of two primers and one probe specific of the
sequence. 18S TaqMan Gene Expression Assay (Hs99999901_s1, Life
technologies) was used as endogenous control and the comparative Ct
method was used for relative quantification of target
expressions.
[0177] Results: The BubR1 mRNA level was increased after the
treatment with the peach flower extract on keratinocytes, compared
to untreated cells. The level was highly significantly (Student's t
test) increased by 46% after 0.1% treatment and by 42% after 0.5%
treatment on keratinocytes. The results are illustrated in FIG.
3.
[0178] Conclusion: The level of BubR1 mRNA was increased in
keratinocytes treated with the peach flower extract for 24
hours.
EXAMPLE 6: BubR1 PROTEIN EXPRESSION STUDY ON KERATINOCYTES AND
FIBROBLASTS TREATED WITH PEACH FLOWER EXTRACT OF EXAMPLE 1
[0179] The object of this study is to determine the influence of
peach flower extract of example 1 on BubR1 protein expression on
cells. To do this, specific labeling by immunofluorescence was
carried out on normal human keratinocytes and fibroblasts.
[0180] Protocol: Treatment solution of peach flower extract were
prepared as in example 2.
[0181] Normal human keratinocytes and normal human fibroblasts were
treated twice with a solution of 0.1% vol/vol or 0.5% vol/vol of
peach flower extract of example 1 for 24 hours. For immunolabelling
by anti-BubR1 antibody, the cells were washed and fixed with
paraformaldehyde at 3.7% for 10 minutes. The cells were then
incubated in the presence of a specific anti-BubR1 antibody (Abcam,
ref. ab4639, mouse monoclonal), and then a secondary suitable
antibody, coupled with a fluorescent dye. After mounting in a
particular medium, the slides were observed by epifluorescence
microscope (Nikon Eclipse 80i microscope). Fluorescence intensity
was quantified by analyzing the image using Volocity 6.3.
software.
[0182] Results: Microscopic observation showed a significantly
highly (Student's t test) increase in BubR1 staining on
keratinocytes and fibroblasts, after the treatment with the peach
flower extract. The results are illustrated in FIGS. 4 and 5,
respectively.
TABLE-US-00006 TABLE 6 Quantification of the microscopic
observations of BubR1 Expression on Keratinocytes & Fibroblasts
Peach flower Peach flower Untreated extract (0.1%) extract (0.5%)
BubR1 expression on 100 112*** 119*** keratinocytes (intensity/cell
area (%)) BubRl expression on 100 107*** 112*** fibroblast
(intensity/ cell area (%))
[0183] Conclusion: The expression of BubR1 protein was increased in
cells treated with the peach flower extract for 24 hours.
EXAMPLE 7: SENESCENT ASSOCIATED BETA-GALACTOSIDASE (SA BETA-GAL)
ACTIVITY STUDY ON FIBROBLASTS TREATED WITH PEACH FLOWER EXTRACT OF
EXAMPLE 1
[0184] The object of this study is to determine the influence of
peach flower extract of example 1 on the beta-galactosidase
senescent marker on cells, after a longer application time.
[0185] Protocol: Treatment solution of peach flower extract were
prepared as in example 2.
[0186] Normal human fibroblasts were treated twice per day with a
solution of 0.1% vol/vol or 0.5% vol/vol of peach flower extract of
example 1, for 12 sub-cultures (P3 to P15) and compared to
untreated fibroblasts at P5. For SA beta-gal activity staining, the
cells were first washed and fixed. They were then incubated
overnight with SA beta-gal stain solution. After mounting in a
particular medium, the slides were observed by light microscopy
(Nikon Eclipse E600 microscope). Blue intensity was quantified by
analyzing the image using Volocity 6.3. software. A normalization
by the number of cells was performed.
[0187] Results: As expected, blue staining increased between
untreated "young" fibroblasts (P5) and untreated senescent
fibroblasts (P15). This increase was statistical and significantly
(Student's test) reduced by 17% and 21% when fibroblasts were
treated with the peach flower extract at 0.1% and 0.5%
respectively.
[0188] Conclusion: The activity of beta-galactosidase senescent
marker was decreased in fibroblasts treated with the peach flower
extract for long term. The results are illustrated in FIG. 6. While
this invention has been described in detail with reference to
certain preferred embodiments, it should be appreciated that the
present invention is not limited to those precise embodiments.
Rather, in view of the present disclosure, which describes the
current best mode for practicing the invention, many modifications
and variations would present themselves to those skilled in the art
without departing from the scope and spirit of this invention.
EXAMPLE 8: SIRT2 ACTIVATION STUDY ON DERMAL FIBROBLASTS TREATED
WITH PEACH FLOWER EXTRACT OF EXAMPLE 1
[0189] The object of this study is to determine the influence of
peach flower extract on Sirtuin 2 cellular migration in human
dermal fibroblasts cells, the testing was performed using extract
of example 1.
[0190] Protocol: Treatment solution of peach flower extract were
prepared by diluting peach flower extract of example 1 at 0.2%
vol/vol, 0.5% vol/vol or 1% vol/vol.
Oris Migration Assay
Materials
[0191] Oris 96 Well Cell Migration Assay System Collagen I Coated
(1-pack), Cat# CMACC1.101 [0192] Cell Stain: [0193] CellTracker
Green Life Technologies 1 mg, Cat# C2925 [0194] Trypsin: [0195]
Trypsin EDTA 1.times. Corning, Cat#25-053-CI [0196] Wash: [0197]
Dulbecco's Phosphate Buffered Saline Solution (DPBS) 1.times., Cat#
25-055-CV [0198] Media: [0199] Dulbecco's Modified Eagle Media
(DMEM) by Life Technologies, Cat# 11965092 [0200] With and without
supplementation [0201] For the supplemented media, media was
supplemented with: [0202] Hyclone Fetal Bovine Serum (FBS)@ 10%,
Cat# SH30071.03
Samples Tested
[0202] [0203] 1. Control: [0204] Media Only [0205] 2. Migration
Positive Control: [0206] PDGF @ 10 ng/ml Sigma Cat# P8147 Lot#
SLBN0763V [0207] 3. CR14031 without preservative "Peach Extract"
(refers to peach flower extract obtained from Example 1) [0208]
Manuf: Ashland Vincience [0209] Code: 865810 [0210] Former Code:
CR14031 [0211] Batch#RM15048 [0212] 0.2% (v/v) [0213] 0.5% (v/v)
[0214] 1% (v/v)
Methods
[0215] Day 1: Cell Seeding of the Oris Migration Plate and
Pre-Treatment with Active:
[0216] Oris Cell Migration assay was removed from 4.degree. C.
refrigeration prior to seeding in order to allow the plate to come
to room temperature.
[0217] Under sterile cell culture conditions, the Oris migration
plate was seeded with 62-year-old passage 11 Normal Dermal Human
Fibroblasts (NHDF's) from Zen Bio Lot# DFM110210B at a density of
20,000 cells per well in 50 ul media per well.
[0218] Then, an equal volume (50 ul) of 2.times. concentrated
treatment in media was added to the designated wells, according to
the following plate layout (n=10), for a final volume in the well
of 100 ul with a resulting lx treatment. The plate was incubated
for 24 hours under standard cell culture conditions of 95%
Humidity, 5% CO.sub.2 and 37.degree. C.
[0219] Shaded wells not utilized and was excess.
[0220] Day 2: Stain cells, Refresh Treatment with Active, Start of
Migration Assay time points.
[0221] The following day, the media and treatments were removed
from the plate, the adherent cells were washed twice in 37.degree.
C. warm Dulbecco's PBS to wash the cells prior to staining.
[0222] Cells were stained using a 10 mM solution of Cell Tracker
Green in serum-free media, and incubated for 30 minutes under
standard cell culture conditions.
[0223] At the end of the 30 min staining, the specialized well
inserts in the Oris Migration assay were removed using the tool
provided, and the CellTracker Green stain was removed.
[0224] The cells were washed twice with pre warmed 37.degree. C.
Dulbecco's PBS to clean away any disturbed cells and remaining
dye.
[0225] 1.times. prepared treatments were refreshed in the plate
according to the provided plate layout and the plate was
immediately read using a fluorescent plate reader at the following
settings: 492 nm ex/517 nm em at the start of the assay designated
t=0. Additional time points at 1, 2, 3, 4, 6, 8, and 24 hours were
taken and data recorded.
[0226] Results: The results show that the treatment with the peach
flower extract increased the cellular migration in 62 yr-old human
dermal fibroblast cells and was concentration dependent. Results
are reported on table:
TABLE-US-00007 TABLE 8 Cellular migration in human dermal
fibroblast cells treated with peach flower extract % Change as
compared to the media control 1 2 3 4 6 8 24 Peach 0.2% 2.21 1.37
1.81 2.33 1.96 1.66 2.63 flower 0.50% 9.04 3.64 3.59 3.65 3.53 2.99
3.82 extract 1% * * * * * * * 10.53 9.41 8.31 8.66 8.75 7.93
8.17
[0227] Conclusion: By supporting Sirt2 through the peach flower
extract, we were able to help cells to regain cellular migration
activity in mature skin cells.
[0228] The results are illustrated in FIG. 7.
EXAMPLE 9: MEASUREMENT OF AVERAGE CELL NUMBER AFTER TREATMENT WITH
PEACH FLOWER EXTRACT OF EXAMPLE 1
[0229] The object of this study is to determine the influence of
peach flower extract on average cell number, the testing was
performed using extract of example 1.
[0230] Protocol: Treatment solution of peach flower extract were
prepared by diluting peach flower extract of example 1 at 0.2%
vol/vol, 0.5% vol/vol or 1% vol/vol.
Alamar Blue Protocol--Assay for Cell Proliferation
Determination
Materials
[0231] 96 well plate [0232] Alamar Blue Solution Invitrogen, Cat#
DAL1100 [0233] Dulbecco's Modified Eagle Media (DMEM) by Life
Technologies, Cat# 11965092 [0234] Supplemented with: [0235]
Hyclone Fetal Bovine Serum (FBS)@10%, Cat# SH30071.03
Methods
[0236] 96 well plate was seeded with 20,000 cells per well of
62-year-old passage 11 Normal Dermal Human Fibroblasts (NHDF's)
from Zen Bio Lot# DFM110210B with treatment as per plate layout
provided, and allowed to culture under normal cell culture
conditions for 48 hours.
[0237] At the end of 48 hours, media and treatment were removed and
a 10% solution of Alamar Blue in media was created, and 100 ul of
this solution was pipetted into all wells of the plate.
[0238] The plate was then returned to the incubator, under standard
cell culture conditions for 1 hour. At the end of the hour
incubation, the absorbance at 570 nm was measured on a plate
reader.
[0239] Results were analyzed by graphing the O.D. at 570 nm vs.
samples tested.
[0240] Results: Alamar blue reading is proportional to the number
of cells that are alive in the plate. By treating with the peach
flower extract, which supports Sirt2, we measured an increase of
cell number in mature human dermal fibroblasts (62 yr-old).
[0241] Conclusion: The support of SIRT2 in mature skin cells is
helping them to proliferate, which makes sense as SIRT2 is related
to the cytoskeleton on the microtubules, which are essential for
cells to divide.
[0242] The results are illustrated in FIG. 8.
TABLE-US-00008 TABLE 9 Average cell number measured through
absorbance @ 570 nm after treatment with peach flower extract Peach
flower Peach flower Peach flower extract (0.2%) extract (0.5%)
extract (1%) Absorbance @ +47%** +73%*** +73%*** 570 nm
* * * * *